Acylated uridine and cytidine and uses thereof

ABSTRACT

The invention relates to compositions comprising acyl derivatives of cytidine and uridine. The invention also relates to methods of treating hepatopathies, diabetes, heart disease, cerebrovascular disorders, Parkinson&#39;s disease, infant respiratory distress syndrome and for enhancement of phospholipid biosynthesis comprising administering the acyl derivatives of the invention to an animal.

This is a Divisional of application Ser. No. 07/997,657, filed Dec. 30,1992, now U.S. Pat. No. 5,470,838 which is a Continuation of Ser. No.07,438,493 filed Jun. 26, 1990, abandoned; which is a CIP of Ser. No.07/115,929 filed Oct. 28, 1987, abandoned.

FIELD OF THE INVENTION

This invention relates generally to acyl derivatives of cytidine anduridine and to the use of those derivatives to deliver exogenousribonucleosides to animal tissue. More specifically, this inventionrelates to the acyl derivatives of cytidine and uridine, and the uses ofthose novel derivatives to deliver these ribonucleosides to animaltissue and thereby to support cellular metabolic functions. Even morespecifically, this invention relates to the use of the novel acylderivatives to treat or prevent a variety of physiological andpathological conditions, including treatment of liver disease or damage,cerebrovascular disorders, respiratory distress syndromes, cardiacdamage, and other clinical conditions.

BACKGROUND OF THE INVENTION

There are many physiological and pathological conditions of animaltissue where the supply of exogenous ribonucleosides may have usefultherapeutic applications. In a number of physiological and pathologicalconditions, the administration to an animal of RNA, nucleotides, orindividual or mixtures of nucleosides, has been shown to improve thenatural repair processes of the affected cells.

There are many important metabolic reactions that are usuallyfunctionally subsaturated and limited by availability of eithersubstrates or cofactors. Such rate-limiting compounds may be eithernutritionally essential or synthesized de novo in the body. Underconditions of tissue trauma, infection or adaptation to physiologicaldemand, particularly when cellular repair or regeneration processes areactivated, the optimum nutritional, biochemical, or hormonal environmentfor promoting such repair may be quite different from the requirementsfor normal cell and tissue function. In such cases, therapeutic benefitmay be derived by providing appropriate conditionally essentialnutrients, such as ribonucleosides or metabolites which may be requiredin quantities not usually available from a normal diet. The therapeuticpotential for this strategy of directly supporting the metabolicfunction of damaged or diseased tissues has not been realized incontemporary medical practice.

At the cellular level of organization, there are specific metabolicresponses to trauma that are involved, in a variety of tissues, in theprocesses of tissue repair, regeneration, or adaptation to alteredfunctional demand. Most processes of tissue damage and repair areaccompanied by a substantial increase in the activity of the hexosemonophosphate pathway of glucose metabolism.

The hexose monophosphate pathway is the route of formation for thepentose sugars (e.g., ribose) which are necessary for nucleotide andnucleic acid synthesis. The availability of ribose is rate limiting fornucleotide synthesis under most physiological or pathologicalconditions. Rapid production of nucleotides for the synthesis of nucleicacids and nucleotide-derived cofactors (such as cytidinedi-phosphocholine (CDP choline) or uridine di-phosphoglucose (UDPG) isessential for the processes of tissue repair and cellular proliferation.Even though nucleotides are synthesized de novo from simpler nutrients,so that there is not an absolute dietary requirement for directnucleotide precursors, many tissues may not have optimal capacity fornucleotide synthesis particularly during tissue repair or cellularproliferation.

It is possible to bypass the limited capacity of the hexosemonophosphate pathway by providing preformed ribonucleosides directly totissues where they are incorporated in the nucleotide pools via the“salvage” pathways of nucleotide synthesis. It is also possible thatpyrimidine ribonucleosides may exert therapeutic influences throughmechanisms unrelated to the support of nucleotide biosynthesis.

The effects of the administration of pryrimidine nucleosides, and inparticular, uridine and cytidine, on a variety of physiological andpathological conditions in experimental animals, isolated tissues, andto some extent, in humans, have been extensively studied. These aresummarized below.

(1) Heart

In isolated rat hearts subjected to low-flow ischemia, reperfusion withuridine induced restoration of myocardial ATP levels, total adeninenucleotide content, uridine nucleotide levels, and glycogen content.Ischemia was reported to produce a breakdown of creatinine phosphate,ATP, uridine nucleotides and glycogen. Aussedat, J., Cardiovasc, Res.17:45-151 (1983).

In a related study, perfusion of isolated rat hearts with uridineresulted in a concentration-dependent elevation of myocardial uracilnucleotide content. Following low-flow ischemia, the rate ofincorporation of uridine was increased twofold. Aussedat, J., et al.,Mol. Physiol. 6:247-256 (1984).

In another study, isoproterenol was administered to rats which depletedcardiac glycogen stores and reduced myocardial UTP and UDP-glucoselevels. Despite the spontaneous restoration of myocardial UTP levels,UDP-glucose concentrations remained depressed unless uridine or ribosewere administered. Prolonged intravenous infusion of ribose or uridineresulted in a restoration of myocardial glycogen. Thus, there may becompartmentation of uridine nucleotides in the heart, with the poolsbeing fed differentially by the salvage of de novo pathways ofpyrimidine synthesis. Aussedat, J., et al., J. Physiol. 78:331-336(1982).

The effects of nucleosides on acute left ventricular failure in isolateddog heart was studied by Buckley, N. M., et al., Circ. Res 7:847-867(1959). Left ventricular failure was induced in isolated dog hearts byincreasing aorta pressure. In this model, guanosine, inosine, uridineand thymidine were found to be positive inotropic agents, while cytidineand adenosine were negatively inotropic.

Sodium uridine monophosphate (UMP) and potassium orotate were found toincrease the animal's resistance to subsequent adrenaline-inducedmyocardial necrosis. These compounds reduced mortality and improvedmyocardial function as assessed by ECG readings, biochemical findings,and relative heart weight. Intravenous administration of UMP exerted amore pronounced prophylactic effect that did potassium orotate.Kuznetsova, L. V., et al., Farmakol.-Toksikol 2:170-173 (1981).

In a study on the effects of hypoxia in isolated rabbit hearts,myocardial performance declined while glucose uptake with glycolysis,glycogenolysis and breakdown of adenine nucleotides were reportedlyincreased. Administration of uridine increased myocardial performance,glucose uptake and glycolysis and also diminished the disappearance ofglycogen and adenine nucleotides from hypoxic hearts. Uridine alsoincreased glucose uptake, glycolysis, levels of ATP and glycogen, aswell as myocardial performance in propranolol-treated hearts. Kypson,J., et al., J. Mol. Cell. Cardiol. 10:545-565 (1978).

In a study of pyrimidine nucleotide synthesis from exogenous cytidine inthe isolated rat heart, myocardial cytosine nucleotide levels weresignificantly increased by a 30 minute supply of cytidine. Most of thecytidine was recovered as part of cytosine nucleotides and uracilnucleotides. Very little of the cytidine that was taken up was convertedinto uridine nucleotides. These results suggest that the uptake ofcytidine can play an important part in myocardial cytosine nucleotidemetabolism. Lortet, S., et al., Basic Res. Cardiol. 81:303-310 (1986).

In another study, myocardial fatigue was produced by repeated, briefligations of the ascending aorta. Intravenous administration of amixture of uridine and inosine after the fifth such ligation temporarilystopped the development of fatigue in the myocardium. Pretreatment withan undisclosed amount of uridine prevented the decrease in maximalpressure upon aortic ligation that is observed 2 hours after aorticstenosis. Meerson, F. C., In: Tr. Vseross. S′ezda Ter., Myasnikov. A. L.(ed.), Meditsina (publisher), Moscow, p. 27-32 (1966).

In another study, the use of glucose and uridine to controlcontractability and extensibility disturbances in the non-ischematizedcompartments of the heart after myocardial infarction were studied. Thedeficits in contractability and extensibility were reported to be due tosustained sympathetic nervous activity. The addition of glucose oruridine in vitro restored contractability and extensibility of theisolated atrial tissue. Meerson, F. Z., et al., Kardiologiya 25:91-93(1985).

Despite the above results which were observed in isolated hearts or insitu organ preparations, the administration of uridine to intact (i.e.,alive and free-running) animals has not been demonstrated to bebeneficial. Thus, while Eliseev, V. V., et al., Khim-Farm. Zh.19:694-696 (1985) (Ca 103:82603k) disclose that uridine-5′-monophosphatehas a protective effect on rats with adrenaline-induced myocardialdystrophy, uridine was found to be relatively ineffective. Moreover,Williams, J. F., et al., Aust. N. Z. J. Med.6:Supp. 2, 60-71(1976),disclose that with rats developing hypertrophy of the heart, there wasno difference between rats which were treated with uridine compared withcontrols. Thus, except for rats which received continuous infusion ofuridine (Aussedat et al., supra), no beneficial effect on pathologyrelated to the heart has been demonstrated with uridine administration.

(2) Muscles

Exposure to uridine has also been found to enhance glucose uptake andglycogen synthesis in isolated skeletal and cardiac muscle. Kypson, J.,et al., Bioch. Pharmacol. 26:1585-1591 (1977). Uridine and inosine werefound to stimulate glucose uptake in isolated rat diaphragm muscle.However, only uridine increased glycogen synthesis. Both nucleosidesinhibited lipolysis in adipose tissue. Kypson, J., et al., J. Pharm.Exp. Ther. 199:565-574 (1976).

(3) Liver

Administration of cytidine and uridine has also been reported to beeffective in enhancing the regeneration of the liver in rats acutelypoisoned with carbon tetrachloride. Bushma, M. I., et al., Bull. Exp.Biol. Med. 88:1480-1483 (1980).

There have been a number of reports relating to the therapeuticadministration of nucleotides and RNA. The beneficial effects of RNA ornucleotides are probably due to their being broken down to individualribonucleosides by phosphatases. For example, injection of cytoplasmicRNA from the rat liver into mice during chronic poisoning with CCl₄reduced the mortality among the animals. Moreover, the number of foci ofnecrosis were reduced and the number of interlobular connective tissuefibers in the liver were increased. An increase in the mitotic activityof the liver cells was also observed. Chernukh, A. M., et al., Bull.Exp. Biol. Med.70:1112-1114 (1970).

Administration of RNA, mixed nucleotides, or hydrocortisone, eitheralone or in various combinations, was found to increasetyrosine-alpha-ketoglutarate activity in rat liver. Administration ofRNA or nucleotides elevated enzymatic activity beyond the level attainedafter hydrocortisone administration alone. The authors speculated thatthe RNA or nucleotides may act via two mechanisms:

first, a nonspecific stress effect, mediated through stimulation ofadrenal steroid release, or secondly, through provision of limitingsubstrates for RNA synthesis. Diamondstone, T. I., et al., Biochim.Biophys. Acta 57:583-587 (1962). In a study on human patients withhepatic cirrhosis, administration of cytidine and uridine improvedinsulin sensitivity in the cirrhotic patient, but had no effect oninsulin sensitivity in patients without liver disease. Ehrlich, H., etal., Metabolism 11:46-55 (1962).

In a study of repair after mechanical trauma in the liver, a rapid,sustained increase in RNA content of cells at the border ofexperimentally induced trauma was observed. DNA concentrations in thetraumatized area began to rise on the third day after injury andcontinued to rise till the 10th day. The diabetic rat liver, incontrast, showed poor RNA and DNA contents. Increases in the tissuecontent of RNA and DNA around the traumatized site were delayed andstrongly depressed relative to nondiabetic livers. The failure of RNAsynthesis, which gives rise to poor wound healing in the diabetic liver,was attributed to deficient activity of the hexose monophosphate pathwayof glucose metabolism as observed in diabetics. Shah, R. V., et al., J.Anim. Morphol. Physiol. 25:193-200 (1978); Shah, R. V., et al., J. Anim.Morphol. Physiol. 21:132-139 (1974).

In another study, the availability of UDPG was found to be rate-limitingfor hepatic glycogen synthesis under some conditions. When culturedhepatocytes were incubated with uridine, there was an increase in theincorporation of glucose into glycogen and tissue uridine nucleotidepools were expanded. When uridine was omitted from the incubationmixture, levels of UTP and UDPG dropped markedly during a 1 hourincubation. Songu, E., et al., Metabolism 30:119-122 (1981). In a studyof patients with alcoholic hepatitis, a beneficial effect ofuridine-disphosphoglucose, when administered intramuscularly orintravenously, was found in biochemical indices as well as physiologicaland psychological symptoms. Thus, pyrimide nucleosides are effective intreatment of some forms of pathology of the liver.

(4) Diabetes

Nucleosides are also useful for the treatment of diabetes. Inexperimental diabetes, RNA synthesis is reduced in a number of tissues.Administration of oral sodium ribonucleate was found to increase therate of RNA biosynthesis in tissues of diabetic rats. Germanyuk, Y. L.,et al., Farmakol. Toksikol. 50-52 (1979). This effect is probably aresult of hydrolysis of the administered RNA to give individualribonucleotides and/or ribonucleosides. The failure of RNA synthesis inthe diabetic rat liver has been attributed to the deficient activity ofthe hexose monophosphate pathway of glucose metabolism in diabetes.Shah, R. V., et al., J. Anim. Morphol. Physiol. 25:193-200 (1978).

(5) Phospholipid Biosynthesis

Cytidine nucleotides have been implicated in phospholipid biosynthesis.For example, Trovarelli, G., et al., Neurochemical Research 9:73-79(1984), disclose that upon the intraventricular administration ofcytidine into the brain of rats, a measurable increase in theconcentrations of all the nucleotides, CDP-choline, CDP-ethanolamine,and CMP occurred. The authors state that the low concentration of freecytidine nucleotides in nervous tissue likely limits the rate ofphospholipid biosynthesis.

(6) Brain

Administration of cytidine and uridine has also been reported to beeffective in the treatment of various neurological conditions inanimals. For example, Dwivedi et al., Toxicol. Appl. Pharmacol. 31:452(1978) disclose that uridine, administered by intraperitoneal injectionin mice, is an effective anticonvulsant, providing strong protectionagainst experimentally-induced seizures.

Geiger et al., J. Neurochem 1:93 (1956) disclose that the functionalcondition of circulation-isolated cat brains perfused with washed bovineerythrocytes suspended in physiological saline remained normal for onlyabout 1 hour. If either the animal's liver was included in the perfusioncircuit, or cytidine and uridine were added to the perfusate, thefunctional condition of the brain remained good for at least 4 to 5hours. The cytidine and uridine tended to normalize cerebralcarbohydrate and phospholipid metabolism. The authors suggest that thebrain is dependent upon a steady supply of cytidine and uridine, whichare perhaps normally supplied by the liver.

Sepe, Minerva Medica 61:5934 (1970), disclose the effect of dailyintramuscular injections of cytidine and uridine in neurologicalpatients, most suffering from cerebrovascular disorders. Beneficialresults were obtained, particularly with respect to restoration of motorfunction, and in improving recovery after cranial trauma. No undesirableside effects were observed.

Jann et al., Minerva Medica 60:2092 (1969) disclose a study of patientswith a variety of neurological disorders which were treated daily withintramuscular injections of cytidine and uridine. Beneficial effectswere observed, particularly in cerebrovascular disorders involving motorfunction and mental efficiency. No undesirable side effects wereobserved.

Monticone et al., Minerva Medica 57:4348 (1966), disclose a study ofpatients with a variety of encephalopathies which were treated withdaily intramuscular injections of cytidine and uridine. Beneficialeffects were found in most patients, particularly those withcerebrovascular disorders or multiple sclerosis. No undesirable sideeffects were observed.

One method that has heretofore been used, in effect, to introducecytidine equivalents into patients is the administration ofcytidine-diphosphocholine (CDP-choline). Cytidine-diphosphocholine, anintermediate in the biosynthesis of phosphatidyl choline (lecithin) isused therapeutically in Europe and Japan (under such names as Somazina,Nicholin, and Citicholine) for treating a variety of disorders.Therapeutic efficacy has been documented in central nervous systempathologies including brain edema, cranial trauma, cerebral ischemia,chronic cerebrovascular diseases, and Parkinson's disease. The mechanismunderlying the pharmacological actions of this compound is believed toinvolve support of phospholipid synthesis, restoration of thebiochemical “energy charge” of the brain, or a possible effect onneurotransmitter (particularly dopamine) function.

Examination of the fate of CDP-choline following its administration toanimals or humans indicates that this compound is very rapidly degraded,yielding cytidine, choline, and phosphate. After oral administration, nointact CD{-choline enters the circulation, although plasma cytidine andcholine concentrations rise. After intravenous injection, breakdown tocytidine and choline occurs within about 30 seconds. Therefore, it isdifficult to attribute the therapeutic effects of exogenous CDP-cholineto the entry of this compound directly into cellular metabolism.

Therapeutic benefits in cerebral pathologies similar to those obtainedwith CDP-choline have been achieved following administration of cytidineand uridine to humans and experimental animals. Therefore, CDP-cholineappears to serve merely as an inefficient, expensive “prodrug” forcytidine, use of which perhaps hinders rather than enhances thetransport of cytidine to target tissues, compared to administration ofcytidine itself. Administration of choline by itself does not result inthe therapeutic benefits obtained after administration of eithercytidine or CDP-choline. It would thus be advantageous to developmethods for delivering cytidine to the brain that are less expensiveand/or more efficient than administration of CDP-choline or cytidineitself.

Uridine-diphosphoglucose, uridine-disphosphoglucuronic acid, and uridinediphosphate also have been shown to improve certain aspects of liverfunction. Since such phosphorylated compounds, as well as CDP-choline,must in general be dephosphorylated before they will enter cells,administration of uridine, or derivatives or uridine, should represent asubstantial improvement, in terms of both efficiency and cost, over theuse of the phosphorylated pyrimidine derivatives.

(7) Immunological System

Cytidine and uridine may also have important influences of the functionof the immune system. Kochergina et al. (Immunologiya 0(5):34-37, 1986)disclose that administration of either cytidine-5′-monophosphate oruridine-5′-monophosphate to mice simultaneously with an antigen (sheepred blood cells) results in a strong enhancement (relative to theresponse in animals treated with only the antigen) of the humoral immuneresponse to a subsequent challenge with the antigen. Enhancedresponsiveness of T-helper lymphocytes was reported to underlie thisphenomenon. Thus, cytidine or uridine may be useful as adjuncts toimprove the efficacy of vaccines, to improve the responsiveness of theimmunce system in an immunocompromised patient, or to modify immuneresponse in experimental animals. Van Buren et al., (Transplantation40:694-697 (1985)) disclose that dietary nucleotides are necessary fornormal T-lymphocyte function; they did not, however, evaluate theinfluence of supra-normal amounts of dietary or parenterallyadministered nucleotides or nucleosides.

In vivo, exogenous uridine itself is catabolized to a large extent,rather than taken up and utilized for nucleotide synthesis. Gasser, T,et al., Science 213:777-778 (1981), disclose that the isolated, perfusedrat liver degrades more than 90% of infused uridine in a single passage.Much of the uridine released by the liver in the portal vein is fromdegradation of liver nucleotides synthesized de novo rather than fromarterial uridine. This accounts for the poor utilization of administereduridine in peripheral tissues.

For example, Klubes, P., et al., Cancer Chemother. Pharmacol. 17:236-240(1986), disclose that after oral administration of 350 (mg/kg of uridinein mice, plasma levels of uridine were not perturbed. In contrast,plasma levels of uracil, a catabolite of uridine, peaked at 50 microthen declined and returned to normal after 4 h. Elevation of plasmauridine levels was observed only after oral administration of high dosesof uridine (3500 mg/kg). However, such doses would be much too high foran adult human since they would amount to about 200 g/dose.

A novel strategy for improving the bioavailability of cytidine oruridine after oral or parenteral administration is to administerderivatives of cytidine or uridine containing particular substituentswhich improve the pharmacokinetic or other pharmaceutical properties(e.g., transport across biological membranes) of these nucleosides.Properly chosen substituents, of which acyl substituents are best) willundergo enzymatic or chemical conversion back into cytidine or uridinefollowing administration.

Certain acylated uridine and cytidine derivatives are known, per se.Honjo, et al., in British Patent No. 1,297,398, describe n⁴,O^(2′),O^(3′), O^(5′)-tetraacylcytidines and a process for their preparation.The acyl substituents are those derived from fatty acids having fromthree to eighteen carbon atoms.

Beranek, et al., Collection Czechslovak Chem. Commun. (vol. 42, 1977),366-369, describe the preparation of 2′, 3′, 5′-tri-O-acetylcytidinehydrochloride from cytidine by reaction with acetyl chloride in aceticacid.

Sasaki, et al., Chem. Pharm. Bull. (vol. 15, 1967), describe theacetylation of cytidine with acetic anhydride to form N⁴-acetylcytidine,5′-O-acetylcytidine and N⁴,5′-O-diacetylcytidine, among other compounds.

U.S. Pat. No. 4,022,963 to Deutsch, describes methods for acetylatingall of the hydroxyl groups in the sugar portion of some nucleosideswhich include uridine, by a process including the addition of excessacetic anhydride.

Samoileva, et al., Bull. Acad. Sci. USSR Div. Chem. Sci. Vol. 30, 1981,p. 1306-1310, disclose a method for synthesizing aminoacyl or peptidylderivatives of cytidine or cytidine monophosphate using insolublepolymeric N-hydroxysuccinimide. N⁴-BOC-alanyl cytidine was prepared. Theaminoacyl derivatives of cytidine were synthesized as probes forstudying the function of nucleases.

Japanese Patent Publications Nos. 51019779 and 81035196 assigned toAsahi Chemical Ind. KK describe methods for preparing N⁴-acyl-cytidinesby reacting cytidine with acid anhydrides derived from fatty acidscontaining 5 to 46 carbon atoms. The products are said to be lipophilicultra-violet absorbing agents and are also useful as starting compoundsin the preparation of anti-tumor agents.

Watanabe, et al., Angew. Chem., Vol. 78, 1986, p. 589 describe methodsfor selective acylation of the N⁴-amino group of cytidine whereinmethanol is used as a solvent and acid anhydride as acylating agent.Compounds prepared were N⁴-acetyl-, N⁴-benzoyl-, andN⁴-butyryl-citidine.

Rees, et al., Tetrahedron Letters, Vol. 29, 1965, p. 2459-2465 disclosemethods for selective acylation of the 2′ position on the ribose moietyof ribonucleosides. Uridine derivatives were prepared including2′-O-acetyluridine, 2′-O-benzyluridine, and 2′,5′-di-O-acetyluridine andother derivatives. The compounds were prepared as intermediates inoligo-ribonucleotide synthesis.

OBJECTS OF THE INVENTION

While certain acylated derivatives of uridine and cytidine are known andwhile the studies summarized above demonstrate that the presence ofuridine and cytidine is important to the amelioration of a variety ofphysiological and pathological conditions and that methods for enhancingthe delivery of uridine and cytidine to animal tissue may provide animportant source of those nucleosides, the art has heretofore failed toprovide methods for introducing uridine and cytidine into animal tissueat rates sufficiently high to reliably produce therapeutic effects.

It is thus a primary object of this invention to identifypharmaceutically acceptable compounds which can be used efficiently todeliver pharmaceutically effective amounts of uridine and/or cytidine ortheir respective derivatives to animal tissue.

It is still a further object of this invention to provide a family ofuridine and cytidine derivatives which can be effectively administeredorally or parenterally and which have no untoward pharmaceuticaleffects.

It is still a further and related object of this invention to provide afamily of uridine and cytidine derivatives which, when administered toan animal, preferably humans, substantially improves the bioavailabilityof cytidine and uridine by enhancing the transport of those nucleosidesacross the gastrointestinal tract, the blood-brain barrier, and otherbiological membranes and which allow sustained delivery of high levelsof these ribonucleosides to animal tissues.

It is still a further and more specific object of this invention toprovide a family of cytidine and uridine derivatives for the treatmentof a variety of disorders including heart, muscle, plasma, liver, bone,diabetic, and neurological conditions.

These and other objects of the invention are achieved through theadministration of novel acyl derivatives of uridine and cytidine.

Broadly, the acyl derivatives of uridine comprise compounds having theformula (I)

wherein R₁, R₂, R₃, and R₄ are the same or different and each ishydrogen or an acyl radical of a metabolite, with the proviso that atleast one of said R substituents is not hydrogen, or a pharmaceuticallyacceptable salt thereof.

In one embodiment, acyl derivatives of uridine are those having theformula (II)

wherein R₁, R₂, and R₃ are the same or different and each is hydrogen oran acyl radical of

(a) an unbranched fatty acid with 5 to 22 carbon atoms,

(b) an amino acid selected from the group consisting of glycine, L-formsof alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine, and ornithine,

(c) a dicarboxylic acid of 3 to 22 carbon atoms, or

(d) a carboxylic acid selected from one or more of the group consistingof glycolic acid, pyruvic acid, lactic acid, enolpyruvic acid, lipoicacid, pantothenic acid, acetoacetic acid, p-aminobenzoic acid,betahydroxybutyric acid, orotic acid, and creatine,

provided that at least one of said substituents R₁, R₂, and R₃ is nothydrogen, and further provided that if any of said substituents R₁, R₂,and R₃ is hydrogen and if said remaining substituents are acyl radicalsof a straight chain fatty acid, then said straight chain fatty acid has8 to 22 carbon atoms, or a pharmaceutically acceptable salt thereof. Inparticular, preferred dicarboxylic acids include succinic, fumaric, andadipic acids. In another embodiment, the objects of the invention arealso achieved with acyl derivatives of uridine comprising compoundshaving the formula (I) above wherein R₄ is not hydrogen.

The objects of the invention are also achieved by administration of acylderivatives of cytidine comprising compounds having the formula (III)

wherein R₁, R₂, R₃, and R₄ are the same or different and each ishydrogen or an acyl radical of a metabolite, with the proviso that atleast one of said R substituents is not hydrogen, or a pharmaceuticallyacceptable salt thereof.

Preferably, the cytidine derivatives are those having formula (III)wherein the R substituents are the same or different and each ishydrogen or an acyl radical derived from a carboxylic acid selected fromone or more of the group consisting of glycolic acid, pyruvic acid,lactic acid, enolpyruvic acid, an amino acid, a fatty acid of 2 to 22carbon atoms, a dicarboxylic acid, lipoic acid, pantothenic acid,acetoacetic acid, p-aminobenzoic acid, betahydroxybutyric acid, oroticacid, and creatine, or a pharmaceutically acceptable salt thereof.Preferred dicarboxylic acids include succinic, fumaric, and adipicacids.

The invention also includes pharmaceutical compositions which compriseone or more of the novel acylated ribonucleosides described abovetogether with a pharmaceutically acceptable carrier. These compositionsmay take the form of tablets, dragees, injectable solutions and otherforms.

Included among the novel pharmaceutical compositions of the inventionare those comprising certain known acyl derivatives of uridine togetherwith a pharmaceutically acceptable carrier. Such compositions include anacyl derivative of uridine having the formula (I) or (II) withsubstituents R₁, R₂, R₃, and R₄ as defined above, or a pharmaceuticallyacceptable salt thereof. Preferred acyl derivatives of uridine include2′,3′,5′-tri-O-acetyl uridine, 2′,3′,5′-tri-O-propionyl uridine, or2′,3′,5′-tri-O-butyryl uridine.

The invention also includes pharmaceutical compositions of certain acylderivatives of cytidine together with pharmaceutically acceptablecarriers. Such acyl derivatives include those having the formula (III)with substituents R₁, R₂, R₃, and R₄ as described above, or apharmaceutically acceptable salt thereof. Preferred acyl derivatives ofcytidine include 2′,3′,5′-tri-O-acetyl cytidine,2′,3′,5′-tri-O-propionyl cytidine, or 2′,3′,5′-tri-O-butyryl cytidine.

It has been found that the delivery of exogenous uridine or cytidine toanimal tissue can be advantageously accomplished by administering to theanimal an effective amount of one or more of the acyl derivativesdescribed above. It has further been found that physiological orpathological conditions of animal tissue may be advantageously treatedby supporting metabolic functions thereof by increasing thebioavailability of uridine or cytidine to that tissue by administeringto an animal an effective amount of an acyl derivative as describedabove.

The invention contemplates the use of these acyl derivatives fortreating a variety of physiological and pathological conditions,including treatment of cardiac insufficiency and myocardial infarction,treatment of liver disease or damage, muscle performance, treatment oflung disorders, diabetes, central nervous system disorders such ascerebrovascular disorders, Parkinson's disease, and senile dementias.The compounds of the invention improve the bioavailability of cytidineand uridine by enhancing the transport of these nucleosides across thegastrointestinal tract and other biological membranes, and prevent theirpremature degradation.

These advantageous uses of the acyl derivatives of the invention areeffected by administering compositions, as described above, of aneffective amount of one or more of these acyl derivatives and apharmaceutically acceptable carrier.

Administration of the acyl derivatives of cytidine and uridine offercertain advantages over administration of the underivatized compounds.The acyl substituents can be selected to increase the lipophilicity ofthe nucleoside, thus improving its transport from the gastrointestinaltract into the bloodstream. The acylated derivatives are effective whenadministered orally. They are resistant to catabolism by nucleosidedeaminases and nucleoside phosphorylases in the intestine, liver, otherorgans, and the bloodstream. Thus, administration of the acylatedderivatives of the invention, either orally or parenterally, allowssustained delivery of high levels of these ribonucleosides to thetissues of an animal.

DESCRIPTION OF THE FIGURES

FIG. 1: this figure shows the basal heart work output for undamaged(received only saline) rats, rats with experimental myocardial damagebut untreated (received only saline) and rats treated withtriacetyluridine (TAU) and triacetylcytidine (TAC) after experimentalmyocardial damage.

FIG. 2: this figure shows the basal left ventricular systolic pressureof control rats, untreated rats, and rats treated with TAU and TAC afterexperimental myocardial damage.

FIG. 3: this figure shows the basal maximum rate of ventricularcontraction of control rats, untreated rats, and rats treated with TAUand TAC after experimental myocardial damage.

FIG. 4: this figure shows the basal maximum rate of ventricularrelaxation of control rats, untreated rats, and rats treated with TAUand TAC after experimental myocardial damage.

FIG. 5: this figure shows the basal heart rate of control rats,untreated rats, and rats treated with TAU and TAC after experimentalmyocardial damage.

FIG. 6: this figure shows the maximum heart work output of control rats,untreated rats, and rats treated with TAU and TAC and norepinephrineafter experimental myocardial damage.

FIG. 7: this figure shows the maximum left ventricular systolic pressureof control rats, untreated rats, and rats treated with TAU and TAC andnorepinephrine after experimental myocardial damage.

FIG. 8: this figure shows the maximum rate of ventricular contraction(maximum) of control rats, untreated rats, and rats treated with TAU andTAC and norepinephrine after experimental myocardial damage.

FIG. 9: this figure shows the maximum rate of ventricular relaxation(maximum) of control rats, untreated rats, and rats treated with TAU andTAC and norepinephrine after experimental myocardial damage.

FIG. 10: this figure shows the heart rate (maximum) of control rats,untreated rats, and rats treated with TAU and TAC and norepinephineafter experimental myocardial damage.

FIG. 11: this figure shows plasma BSP clearance in rats with liverdamage treated with TAC and TAU or with water (control).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definition of Terms

A “metabolite” is a chemical compound that is formed by, or participatesin, a metabolic reaction. In the context of this application,metabolites refer, in particular, to carboxylic acids known to besynthesized within the human body, and also naturally occurring (butperhaps synthesized rather than extracted) substituents that might bederived from other animal or plant sources. The limiting criteria arethat the compound should be substantially nontoxic and biocompatible,and should readily enter into metabolic pathways in vivo, so as topresent essentially no toxicity during long-term consumption in thedoses proposed. It is preferable that the substituents be metabolizedrather than excreted intact (or conjugated through detoxificationreactions), as concentration of carboxylic acids within the kidney maylead to undesirable excessive acidity. Therefore, carboxylic acids thatnormally or easily participate in intermediary, catabolic, or anabolicmetabolism are preferred substituents. Preferably such carboxylic acidsare of molecular weight less than 1000 Daltons.

“Pharmaceutically acceptable salts” means salts with pharmaceuticallyacceptable acid addition salts of the nucleoside derivatives of theinvention. Such acceptable acids include, but are not limited to,sulfuric, hydrochloric, or phosphoric acids.

“Coadministered” means that each of at least two acyl nucleosidederivatives are administered during a time frame wherein the respectiveperiods of pharmacological activity overlap.

“Acyl derivatives” are derivatives of cytidine or uridine in which asubstantially nontoxic organic acyl substituent derived from acarboxylic acid is attached to one or more of the free hydroxyl groupsof the ribose moiety of cytidine or uridine with an ester linkage,and/or where such a substituent is attached to a primary or secondaryamine in the pyrimidine ring of cytidine or uridine, with an amidelinkage. Such acyl substituents include, but are not limited to, thosederived from acetic acid, fatty acids, amino acids, lipoic acid,glycolic acid, lactic acid, enolpyruvic acid, pyruvic acid, orotic acid,acetoacetic acid, beta-hydroxybutyric acid, creatinic acid, succinicacid, fumaric acid, adipic acid, and p-aminobenzoic acid. Preferred acylsubstituents are compounds which are normally present in the body,either as dietary constituents or as intermediary metabolites, and whichare essentially nontoxic when cleaved from the ribonucleoside in vivo.

“Fatty acids” are aliphatic carboxylic acids having 2 to 22 carbonatoms. Such fatty acids may be saturated, partially saturated orpolyunsaturated.

“Amino acids” include, but are not limited to, glycine, the L forms ofalanine, valine, leucine, isoleucine, phenylalanine, tyrosine, proline,hydroxyproline, serine, threonine, cysteine, cystine, methionine,tryptophan, aspartic acid, glutamic acid, arginine, lysine, histidine,ornithine, and hydroxylysine. However, the invention is not so limited,it being within the contemplation of the invention to include othernaturally occurring amino acids.

The lipophilic acyl derivatives of uridine and cytidine are useful forenhancing the transport of the nucleotides across the gastrointestinaltract in animals. Foremost among such animals are humans. However, theinvention is not intended to be so limited, it being contemplated thatall animals may be treated with the acyl derivatives of the presentinvention with attendant beneficial effect.

Although the invention is not bound by a specific mechanism of action,the compounds of the present invention appear to effect their beneficialactivity by increasing the bioavailability of cytidine and uridine, andthereby, improving tissue regeneration, repair, performance, resistanceto damage, and adaptation to physiological demand. They may work, aswell, by increasing the bioavailability of nucleoside anabolites, e.g.,nucleotides or nucleotide-derived cofactors. Administration of thenucleosides per se increases their bioavailability but, due to rapidcatabolism, this may not result in significant elevation of nucleotidelevels; i.e., one doesn't necessarily get an increase in plasma levelsbecause at lower nucleoside levels there is rapid uptake by the cellswhereas at higher levels there is saturation and the excess is degraded.The invention is believed to work by delivering a sustained supply ofnucleoside at lower levels.

Preferred acyl derivatives of cytidine or uridine for enhancingtransport across biological membranes are those which are morelipophilic than are the parent nucleosides. In general, lipophilic acylnucleoside derivatives have acyl substituents which are nonpolar (asidefrom the carboxylate group). Such acyl substituents are derived fromacids including, but not limited to, acetic acid, lipoic acid, and fattyacids. One of ordinary skill in the art can determine whether aparticular acyl nucleoside derivative is more lipophilic than theunderivatized nucleoside using standard techniques, i.e., comparison ofthe partition coefficients determined in water-octanol mixtures.Following passage of the acylated nucleoside derivative from thegastrointestinal tract into the bloodstream or across other biologicalmembranes, the acyl substituents are cleaved by plasma and tissueesterases (or amidases) to give the free nucleosides.

The rate of removal of the acyl substituents in vivo is a function ofthe specificity of plasma and tissue deacylating enzymes (primarilyesterases or amidases). Acyl substituents attached to an amine group inthe pyrimidine ring of cytidine or uridine with an amide linkage arecleaved more slowly than are substituents attached to hydroxyl groups ofribose with an ester linkage.

It is also possible to prepare acyl nucleoside derivatives which containpolar and nonpolar acyl substituents. The polar acyl substituent willretard passage of the nucleoside derivative from the gastrointestinaltract, allowing for a more sustained delivery of the compound into thebloodstream after a single dose. The polar group may be cleaved byesterases, amidases, or peptidases present in the intestinal tract togive a nucleoside with a nonpolar acyl substituent which may thenefficiently enter the circulation. Polar acyl substituents may be chosenby one of ordinary skill in the art, without undue experimentation,which are cleaved at a faster rate than are nonpolar acyl substituents.

The acyl derivatives are also less susceptible to degradation of thenucleoside moiety by enzymes in plasma and non-target tissues, and arealso less susceptible to elimination from the bloodstream via thekidneys. For parenteral injection, acyl derivatives with polar acylsubstituents, which are therefore water soluble yet resistant topremature degradation or elimination, may be used with advantage.Preferred acyl derivatives in such application include glycolate andlactate and those derived from amino acids with polar side chains.

Therapeutic Uses

Administration of the acyl derivatives of cytidine may be useful intreating lung disorders, including infant respiratory distress syndrome(IRDS), and in metabolic disorders that affect pulmonary function. Theacyl derivatives appear to support or enhance phospholipid biosynthesisand surfactant formation in the lung. The main component of thesurfactant, phosphatidyl choline, is derived from cytidinediphosphocholine. Thus, administration of the acylated form of cytidinewill support or augment the capacity of pneumocytes to synthesizephospholipids and generate surfactant. The beneficial effects ofcytidine acyl derivatives may be enhanced by coadministering uridineacyl derivatives.

In addition, administration of the acyl derivative of cytidine may beuseful in treatment of neural disorders. The acyl derivatives may exerttheir activity by restoring or maintaining brain phospholipidcomposition during or after a period of cerebral hypoxia or stroke.Administration of the acyl derivative of cytidine may also be useful inslowing the onset or progression of degenerative disorders. Disorderssuch as cerebrovascular disorders, Parkinson's disease, and cerebralataxia have been linked to phospholipid levels. The acyl derivatives ofuridine may be advantageously coadministered with the acyl derivative ofcytidine to enhance its effect.

Administration of the acyl derivatives of cytidine and uridine may beeffective for the treatment of cerebrovascular dementias and Parkinson'sdisease. Cerebrovascular dementias and Parkinson's disease causegradual, generally symmetric, relentlessly progressive wasting away ofthe neurons. Cerebral ataxia is characterized by loss of nerve cellsprincipally affecting the Purkinje cells.

Therefore, administration of the acyl derivatives of cytidine anduridine may exert their activity by enhancing phospholipid biosynthesisand thereby ameliorating the effects of cerebrovascular disorders,Parkinson's disease and cerebral ataxia.

The invention also relates to treatment of physiological orpathophysiological conditions where the body's capacity to synthesizenucleic acids is suboptimal. These conditions include diabetes,senescence, and adrenal insufficiency. Administration of the acylderivatives of cytidine and uridine may provide a beneficial effect byproviding a sustained delivery of high levels of cytidine and uridine togive sufficient pools of nucleotides which are necessary for thebiosynthesis of enzymes crucial for cellular self regeneration.

Although the invention is not bound by any one mode of action, thecompositions of the present invention, in and of themselves, appear toact by enhancing nucleotide and nucleic acid synthesis and proteinsynthesis by providing nucleosides under conditions wherein de novosynthesis is not sufficient for supporting optimal rates of nucleotideand nucleic acid synthesis. Thus, the compounds may find utility intreatment of cardiac insufficiency, myocardial infarction, liver diseaseincluding cirrhosis, and by reversing the pathological effects ofdiabetes by accelerating nucleic acid synthesis and thereby proteinsynthesis.

The acyl derivatives of uridine and cytidine may be administered toimprove ventricular function after myocardial infarction or in treatingor preventing cardiac insufficiency. The acyl nucleoside derivatives ofthe invention, which appear to support the cellular mechanisms involvedin calcium sequestration, and which thereby preserve or support cellularATP regeneration, may have significant therapeutic value in preventingor treating some of the deleterious effects of myocardial insults.

The compositions of the present invention may be coadministered withdrugs which are used to treat cardiac insufficiency, e.g., digitalis,diuretics and catecholamines.

It is possible to attenuate load-induced myocardial damage, and topromote stable hyperfunction by providing to the heart substances thatsupport the biochemical processes involved in calcium sequestration andRNA biosynthesis. Uridine and cytidine are useful compounds in thiscontext. Uridine has been reported to be relatively ineffective insupporting myocardial hyperfunction in vivo; however, this may bebecause uridine is rapidly degraded by plasma and tissue enzymes, whichconsequently prevents its utilization by the heart. The invention isbased partly on the finding that it is possible to improve delivery ofuridine to the heart by administering the acyl derivatives whichgradually release free uridine into the bloodstream over an extendedperiod of time.

The acyl derivatives of uridine may be administered to treat hypoxia oranoxia. These acyl derivatives appear to act by enhancing biosynthesisof uridine diphosphoglucose, a necessary intermediate in glycogensynthesis, to improve tissue resistance to hypoxia or anoxia andpreserve the functional capacity of tissues, in particular cardiac.Uridine acyl derivatives may be used for the treatment of hypoxia,anoxia, ischemia, excessive catechol-aminergic stimulation, and digoxintoxicity.

The compounds of the present invention may also find utility incountering some of the long term complications of diabetes, whichinclude neuropathies, arteriopathies, increased susceptibility to bothcoronary arteriosclerosis and myocardial infarction, and blindness.Since de novo nucleotide synthesis is suppressed in diabetes, exogenousacyl derivatives of nucleosides have therapeutuc value in treatingdiabetes. In addition, the derivatized forms of the nucleosides may beadministered to provide sufficient pools of nucleosides necessary forthe biosynthesis of enzymes crucial for cellular self regeneration.Thus, the invention also relates to methods for treating, for example,diabetic liver, or vascular disease by administering the acyl nucleosidederivatives of the invention. The acyl nucleoside derivatives are alsouseful in supporting or enhancing muscular hypertrophy or hyperfunctionin response to increased demand. Such demand may occur after sustainedmuscular exertion.

Preferred acyl substituents include acetyl, propionyl, and butyrylgroups. Preferred acyl nucleoside derivatives include2′,3′,5′-tri-O-acetyl cytidine, 2′,3′,5′-tri-O-acetyl uridine,2′,3′,5′-tri-O-propionyl uridine, 2′,3′,5′-tri-O-propionyl cytidine,2′,3′,5′-tri-O-butyryl cytidine and 2′,3′,5′-tri-O-butyryl uridine. Itcan be advantageous to coadminister acyl derivatives of both cytidineand uridine.

Typical dosage forms are equivalent to 10 to 3000 mg of cytidine and/oruridine in the form of their acyl derivatives or the pharmaceuticallyacceptable salt thereof, 1 to 3 times per day. This corresponds to, forexample, 15 to 4500 mg of 2′,3′,5′-tri-O-acetyl cytidine and2′,3′,5′-tri-O-acetyl uridine.

For treatment of cardiac insufficiency, myocardial infarction and theconsequences of hypertension, a composition comprising 25 to 100 molepercent of the acyl derivative of uridine may be coadmnistered togetherwith 75 to 0 mole percent of the acyl derivative of cytidine with theproviso that the amounts of the acyl derivatives of cytidine and uridinedo not exceed 100 mole percent. For example, 1125-4500 mg of2′,3′,5′-tri-O-acetyluridine may be administered with 0-3475 mg of2′,3′,5′-tri-O-acetylcytidine.

For treatment of cerebrovascular disorders, diabetes, liver damage anddisease, and to increase muscle performance, a composition comprising 25to 75 mole percent of the acyl derivative of uridine may becoadministered together with 75 to 25 mole percent of the acylderivative of cytidine with the proviso that the amount of the acylderivatives of uridine and cytidine do not exceed 100 mole percent. Forexample, 1125-3375 mg of 2′,3′,5′-tri-O-acetyluridine may becoadministered with 1125-3375 mg of 2′,3′,5′-tri-O-acetyl cytidine.

For treatment of respiratory distress syndrome, 25 to 100 mole percentof the acyl derivative of cytidine may be coadministered with 75 to 0mole percent of the acyl derivative of uridine with the proviso that theamounts of the acyl derivatives of uridine and cytidine do not exceed100 mole percent. For example, 1125-4500 mg of 2′,3′,5′-tri-O-acetylcytidine may be coadministered together with 0-3375 mg of2′,3′,5′-tri-O-acetyl uridine.

Examples of Therapeutic Administration

Cardiac Insufficiency

Acyl derivatives of cytidine and uridine are useful in the treatment ofseveral varieties of cardiac insufficiency. They are effective insupporting sustained compensatory hyperfunction in the case of increasedload upon the heart in hypertension, for example, or especially insupporting the function of the surviving portions of the heart after amyocardial infarction. In this latter situation, a mixture of acylderivatives of cytidine and uridine may be given as soon after the onsetof the infarction as possible, followed by chronic oral administrationof a suitable formulation of acyl derivatives of these nucleosides indoses of approximately 0.5 to 3.0 grams per day of each. These compoundsmay be used advantageously in conjunction with conventional treatmentsof myocardial infarction. The nucleoside derivatives have the uniqueadvantage of protecting the heart against damage secondary to overload,hypoxia, or catecholamines without reducing the functional capacity ofthe heart, since they act by enhancing the metabolic integrity of themyocardium, in particular by improving calcium handling. The nucleosidederivatives may also be administered prophylactically to patients atrisk for myocardial infarction or cardiac insufficiency.

For treatment of chronic cardiac insufficiency, which leads tocongestive heart failure, acyl derivatives of cytidine and uridine maybe administered orally in doses ranging from 0.5 to 3 grams per day ofeach nucleoside. The nucleosides may be used in conjunction with otheragents such as digitalis derivatives or diuretics. In addition toimproving myocardial function directly, the nucleoside derivativesreduce digitalis toxicity without impairing its clinical efficacy.

Diabetes

In many tissues of diabetic subjects, cellular pyrimidine nucleotidelevels are reduced; this may contribute to some of the long-termcomplications of diabetes, including arteriopathies, neuropathies, anddecreased resistance of the myocardium to mechanical or biochemicalstress. These complications are related to malfunctions in tissuecalcium handling, in which pyrimidine nucleotides play key roles. It hasbeen reported that daily intramuscular injection of cytidine and uridinereverses the depression in peripheral nerve conduction velocity indiabetic humans (C. Serra, Rif. Med. 85:1544 [1971]). It is preferableto administer acyl derivatives of cytidine and uridine orally insuitable formulations. Doses equivalent to 0.5 to 3 grams of cytidineand uridine are administered daily, in conjunction with conventionalanti-diabetic treatments. The nucleoside derivatives are particularlyuseful in non-insulin dependent diabetes.

Neurological Disorders

In the treatment of the consequences of cerebrovascular disorders, e.g.,stroke and chronic or acute cerebrovascular insufficiency, acylderivatives of cytidine and uridine, particularly those formulated topass through the blood-brain barrier after oral administration, may beadministered in oral doses ranging from 0.5 to 3.0 grams of eachnucleoside per day for at least several months.

In Parkinson's disease, acyl cytidine derivatives are particularlyuseful, and may be given in conjunction with the conventional treatmentof choice, L-DOPA. The cytidine derivatives, administered in oral dosesof 0.5 to 3.0 grams per day, may permit satisfactory clinicalmaintenance on reduced dosages of L-DOPA, which is advantageous becauseL-DOPA has undesirable side effects.

Methods of Preparation of the Compounds

The acyl derivatives of the invention may be prepared by the followinggeneral methods. When the acyl substituent has groups which interferewith the acylation reactions, e.g., hydroxyl or amino groups, thesegroups may be blocked with protecting groups, e.g., t-butyldimethylsilylesters or t-BOC groups, respectively, before preparation of theanhydride. For example, lactic acid may be converted to2-(t-butyldimethylsiloxy)propionic acid witht-butyldimethylchlorosilane, followed by hydrolysis of the resultingsilyl ester with aqueous base. The anhydride may be formed by reactingthe protected acid with DCC.

In the case of amino acids, the N-t-BOC derivative may be prepared,using standard techniques, which is then converted to the anhydride withDCC.

Derivatives containing acyl substituents with more than one carboxylategroup (e.g., succinate, fumarate, or adipate) are prepared by reactingthe acid anhydride of the desired dicarboxylic acid with a2′-deoxyribonucleoside in pyridine.

For example, the 2′,3′,5′-tri-O-acyl derivatives of uridine may beprepared by a modified procedure disclosed by Nishizawa et al., Biochem.Pharmacol. 14:1605 (1965). To one equivalent of uridine in pyridine isadded 3.1 equivalents of an acid anhydride (acetic anhydride, butyricanhydride, etc.), and the mixture heated to 80-85° C. The triacylderivative may then be isolated using standard techniques.Alternatively, uridine may be treated with 3.1 equivalents of a desiredacid chloride (acetyl chloride, palmitoyl chloride, etc.) in pyridine atroom temperature (See Example V).

The 5′acyl derivative of uridine may be prepared according to Nishizawaet al. by reacting uridine with 1 equivalent of the acid anhydride ofthe desired acyl compound in pyridine at room temperature. The reactionis then heated to 80-85° C. for two hours, cooled, and the 5′-acylderivative isolated by standard techniques and purified bychromatography. Alternatively, the 5′-acyl derivative of uridine may beprepared by treating uridine, in pyridine and DMF at 0° C., with 1equivalent of the acid chloride derived from the desired acyl compound.The 5′-acyl derivative of uridine may then be isolated by standardtechniques and purified by chromatography (see Example VI).

The 2′,3′-diacyl derivatives of uridine may be prepared by a procedureadapted from Baker et al., J. Med. Chem. 22:273 (1979). The 5′-hydroxylgroup is selectively protected with 1.2 equivalents oft-butyldimethylsilyl chloride in DMF containing imidazole, at roomtemperature. The 5′-t-butyldimethylsilyl derivative of uridine isisolated by standard techniques, then treated with 2.1 equivalents ofthe acid anhydride of the desired acyl compound in pyridine at 0-5° C.The resulting 5′-t-butyl dimethylsiloxy-2′,3′-diacyl uridine is thentreated with tetrabutylammonium fluoride and the 2′,3′-diacyl derivativeof uridine is isolated by standard techniques (see Example VII).

The secondary amine of 2′,3′,5′tri-O-acyl uridine may then be acylatedaccording to Fujii et al., U.S. Pat. No. 4,425,335, which involvestreatment with 1.1 equivalents of an acid chloride in an aprotic solventcontaining 1-5 equivalents of an organic base, e.g., aromatic aminessuch as pyridine, trialkylamines, or N,N-dialkylanilines. Using thisprocedure, a tetraacyl derivative of uridine may be prepared which hasan acyl substituent on the amino group which is different from the acylsubstituents at the 2′,3′ and 5′ hydroxy groups (see Example VIII).

The secondary amine of 2′,3′,5′tri-O-acyl uridine may then be acylatedaccording to Fujii et al., U.S. Pat. No. 4,425,335, which involvestreatment with 1.1 equivalents of an acid chloride in an aprotic solventcontaining 1-5 equivalents of an organic base, e.g., aromatic aminessuch as pyridine, trialkylamines, or N,N-dialkylanilines. Using thisprocedure, a tetraacyl derivative of uridine may be prepared which hasan acyl substituent on the amino group which is different from the acylsubstituents at the 2′,3′ and 5′ hydroxy groups (see Example VIII).

The 2′,3′,5′tri-O-acyl derivatives of cytidine may be prepared accordingto a method adapted from Gish et al., J. Med. Chem. 14:1159 (1971). Forexample, cytidine hydrochloride may be treated with 3.1 equivalents ofthe desired acid chloride in DMF. The 2′,3′,5′tri-O-acyl derivative maythen be isolated using standard techniques (see Example IX).

The 5′-acyl derivative of cytidine may be prepared according to Gish etal., supra, by treatment of cytidine hydrochloride with 1.1 equivalentsof an acid chloride in DMF, followed by isolation of 5′-acyl cytidine bystandard techniques (see Example X).

Selective acylation of the N⁴-amine of cytidine accomplished accordingto the procedure disclosed by Sasaki et al., Chem. Pharm. Bull. 15:894(1967). This involves treatment of cytidine with 1.5 equivalents of anacid anhydride in pyridine and DMF. The N⁴-acyl derivative of cytidinemay then be isolated by standard techniques (see Example XI).

Alternatively, the N⁴-acyl derivative of cytidine may be prepared bytreatment of cytidine with an acyl anhydride in pyridine or a mixture ofpyridine and DMF. Another procedure for the selective preparation ofN⁴-acyl cytidine involves selective acylation with an acid anhydride ina water-water miscible solvent system according to Akiyama et al., Chem.Pharm. Bull. 26:981 (1978) (see Example XI).

Tetraacyl cytidine derivatives, where all the acyl groups are the same,may be prepared by treating cytidine with at least 4 molar equivalentsof an acid anhydride in pyridine at room temperature. The tetraacylcytidine may then be isolated using standard techniques (see ExampleXII).

To prepare compounds in which the acyl substituent on the N⁴ amino groupis different from the acyl substituents on the hydroxyl groups of theribose ring (e.g., N⁴-palmitoyl 2′,3′,5′tri-O-acetyl cytidine), thedesired acyl substituent is selectively attached to the N⁴ amino groupas described above, and then the hydroxyl groups are acylated with theirintended substituents. Alternatively, the substituents on the ribosemoiety may be attached prior to attachment of the substituent of the N⁴amino group, again using methods described above.

Compositions within the scope of this invention include all compositionswherein each of the components thereof is contained in an amounteffective to achieve its intended purpose. Thus, the compositions of theinvention may contain one or more acyl nucleoside derivatives of uridineor cytidine in amounts sufficient to result, upon administration, inincreased plasma or tissue levels of cytidine or uridine and the acylderivatives thereof, which thereby produce their desired effect.

In addition to the pharmacologically active compounds, the newpharmaceutical preparations may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into the preparationswhich may be used pharmaceutically. Preferably, the preparations,particularly those preparations which can be administered orally andwhich can be used for the preferred type of administration, such astablets, dragees, and capsules and also preparations which can beadministered rectally, such as suppositories, as well as suitablesolutions for administration by injection or orally, contain about from0.1 to 99%, preferably from about 10-90%, of the active compound(s),together with the excipient.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compound(s) with solidexcipients, optionally grinding a resulting mixture and processing themixture of granules, after adding suitable auxiliaries, if desired ornecessary, to obtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as sugars, forexample lactose or sucrose, mannitol or sorbitol, cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phos phate, as well as binders such as starch paste, using, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxypro pylmethyl cellulose,sodium carboxymethyl cellulose, and/or polyvinyl pyrrolidone. Ifdesired, disintegrating agents may be added such as the above-mentionedstarches and also carboxymethylstarch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodiumalginate. Auxiliaries are, above all, flow-regulating agents andlubricants, for example, silica, talc, stearic acid or salts thereof,such as magnesium stearate or calcium sterate, and/or polyethyleneglycol. Dragee cores are provided with suitable coatings which, ifdesired, are resistant to gastric juices. For this purpose, concentratedsugar solutions may be used, which may optionally contain gum arabic,talc, polyvinyl pyrrolidone, polyethylene glycol and/or titaniumdioxide, lacquer solutions and suitable organic solvents or solventmixtures. In order to produce coatings resistant to gastric juices,solutions of suitable cellulose preparations such as acetylcellulosephthalate or hydroxylpropylmethylcellulose phthalate are used. Dyestuffs or pigments may be added to the tablets or dragee coatings, forexample, for identification or in order to characterize differentcombinations of compound doses. Other pharmaceutical preparations whichcan be used orally include push-fit capsules made of gelatin, as well assoft-sealed capsules made of gelatin and a plasticizer such as glycerolor sorbitol. The push-fit capsules contain the active compound(s) in theform of granules which may be mixed with fillers such as lactose,binders such as starches and/or lubricants such as talc or magnesiumsterate, and, optionally, stabilizers. In soft capsules, the activecompounds are preferably dissolved or suspended in suitable liquids suchas fatty oils, liquid paraffin, or polyethylene glycols. In addition,stabilizers may be added.

Possible pharmaceutical preparations which can be used rectally include,for example suppositories which consist of a combination of activecompounds with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, paraffin hydrocarbons,polyethylene glycols or higher alkanols. In addition, it is alsopossible to use gelatin rectal capsules which consist of a combinationof the active compounds with a base. Possible base materials include,for example, liquid triglycerides, polyethylene glycols, or paraffinhydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water soluble form, for example,water soluble salts. In addition, suspensions of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may include substanceswhich increase the viscosity of the suspension which include, forexample, sodium carboxymethylcellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stablizers.

The following examples are illustrative, but not limiting, of themethods and compositions of the present invention. Other suitablemodifications and adaptations of the variety of conditions andparameters normally encountered in clinical therapy and which areobvious to those skilled in the art and are within the spirit and scopeof this invention.

EXAMPLES Example I

Comparison of the Bioavailability of Uridine and Acyl UridineDerivatives in Rats

Silastic catheters were implanted into the right jugular veins ofanesthetized male F344 rats (Retired Breeders, 450-500 grams). Threedays later, blood samples were withdrawn without disturbing the rats.Basal blood samples were taken and the animals were divided into fourgroups, each containing four rats. Each group received a different oneof each of the following compounds; uridine, 2′,3′,5′tri-O-acetyluridine, cytidine, or 2′,3′,5′tri-O-acetyl cytidine. The compounds weregiven in equimolar dosages (0.28 moles/kg) by intubation into thestomach. At intervals of 0.5, 1, 2, 3, and 4 hours after administration,blood samples (0.3 ml) were withdrawn and processed for subsequent assayof cytidine or uridine content by HPLC. In the rat, plasma levels ofuridine were significantly higher (5 to 10 fold) for at least four hoursfollowing ingestion of tri-O-acetyl uridine than after ingestion of anequimolar dose of uridine.

Example II

Comparison of the Bioavailability of Uridine and Acyl UridineDerivatives in Humans

After an overnight fast, a basal venous blood sample was withdrawn froma human subject and then 0.76 moles/kg (28 mg/kg—2 grams in a 70 kgsubject) of tri-O-acetyl uridine was ingested along with 100 ml ofwater. Blood samples (0.5 ml) were withdrawn at intervals of 1, 2, 3,and 4 hours after ingestion of the compound and were processed forsubsequent determination of plasma uridine content by HPLC. On aseparate day, the same procedure was carried out, except that an equalmolar dose (18 mg/kg—1.3 grams in a 70 kilogram subject) of uridine wasingested instead of the acyl derivative. The plasma level of uridine wassubstantially higher following ingestion of tri-O-acetyl uridine thanafter ingestion of the equal molar dose of uridine. Uridine levels weresustained in the useful therapeutic range (greater than 10 micromolar)for at least four hours after oral administration of tri-O-acetyluridine. After administration of oral uridine, plasma levels of thenucleoside exceeded 10 micromolar at only one point in time (two hours).

Example III

Restoration of Depressed Myocardial Function with Acylated PyrimidineRibonucleosides

The experiment described within this example was designed to determinewhether providing exogenous triacetyl uridine and triacetyl cytidinecould help to restore pump function in the ventricular myocardium afterexperimental depression of ventricular function.

Experimental myocardial damage was induced in anesthetized (Nembutal, 50mg/kg i.p.) male F344 rats (250 grams) by constricting the abdominalaorta to an internal diameter of 0.67 mm, followed by injection of asingle dose of isoproterenol hydrochloride (5 mg/kg s.c.). A mixture oftriacetyl cytidine and triacetyl uridine (590 mg/kg of each) wasadministered immediately after, and again 1 hour and 20 hours afteraorta constriction and administration of isoproterenol. Some animalsreceived injections of saline instead of the acetylated nucleosides(untreated), and a group of animals also received saline injections butwere not subjected to aorta constriction or treatment with isoproterenol(controls). Ventricular function was determined 24 hours after aorticconstriction. Animals were anesthetized with sodium pentobarbital (50mg/kg i.p.), and a catheter was implanted in the right jugular vein foradministration of norepinephrine. A second catheter (Intramedic PE-50)was inserted into the left ventricle of the heart via the right carotidartery. Left ventricular systolic pressure (LVSP), the maximum rate ofventricular contraction and relaxation (+dP/dT and −dP/dT, respectively)and heart rate (HR) were measured directly via this catheter, using aStatham-type pressure transducer interfaced to a Stoelting PhysioscribeII polygraph. Values of these parameters were recorded, before and afteri.v. administration of 0.1 ml of norepinephrine bitartrate atconcentrations of 10⁻⁶, 10⁻⁵, and 10⁻⁴M. Electrocardiograms were alsorecorded with this apparatus, using stainless steel needle electrodesinserted subcutaneously in the forelimbs. Heart work output wascalculated as the product of ventricular systolic pressure and heartrate.

Aorta constriction in conjunction with isoproterenol administrationresulted in substantial decrements in myocardial performance compared tointact controls. Left ventricular systolic pressure, +dP/dT, −dP/dT, andheart work output were all significantly depressed (Table 1; FIGS. 1-4).In the animals that received acetylated pyrimidine nucleosides afteraorta constriction and administration of isoproterenol, all of theseparameters were significantly restored toward normal, compared toanimals treated only with isoproterenol (FIGS. 1-4). Heart rate was alsodepressed after experimental myocardial damage (FIG. 5).

TABLE 1 Basal Heart Performance LVSP HR +dP/dT −dP/dT HR × LVSPTREATMENT (mmHg) (bpm) (mmHg/sec) (mmHg/sec) (mmHg/min) Control 141 ±11  386 ± 46 6000 ± 348  5640 ± 528  55,766 ± 10,407 AC + Saline 107 ±14* 283 ± 44 4080 ± 600* 3120 ± 840* 32,633 ± 9,115* AC + TAU&TAC 158 ±9  398 ± 28 6000 ± 480  5640 ± 300  63,518 ± 6,624  *= Significantlydifferent from control value (P .02) Abbreviations: AC = Aortaconstriction + isoproterenol TAU = Triacetyl uridine TAC = Triacetylcytidine LVSP = Left ventricular systolic pressure HR = Heart rate+dP/dT = Maximum rate of ventricular contraction −dP/dT = Maximum rateof ventricular relaxation

TABLE 1 Basal Heart Performance LVSP HR +dP/dT −dP/dT HR × LVSPTREATMENT (mmHg) (bpm) (mmHg/sec) (mmHg/sec) (mmHg/min) Control 141 ±11  386 ± 46 6000 ± 348  5640 ± 528  55,766 ± 10,407 AC + Saline 107 ±14* 283 ± 44 4080 ± 600* 3120 ± 840* 32,633 ± 9,115* AC + TAU&TAC 158 ±9  398 ± 28 6000 ± 480  5640 ± 300  63,518 ± 6,624  *= Significantlydifferent from control value (P .02) Abbreviations: AC = Aortaconstriction + isoproterenol TAU = Triacetyl uridine TAC = Triacetylcytidine LVSP = Left ventricular systolic pressure HR = Heart rate+dP/dT = Maximum rate of ventricular contraction −dP/dT = Maximum rateof ventricular relaxation

The parameters of myocardial performance were monitored in the same ratsfollowing administration of 0.1 ml of 10⁻⁴ M norepinephrine bitartrate.These values represent the maximal performance of the heart, and aredisplayed in Table 2 and FIGS. 6-10.

Discussion

Providing exogenous nucleosides to the myocardium by administering theacyl nucleoside derivatives of the invention prevents or alleviates theimpairments in myocardial performance that normally accompany cardiachyperfunction and hypertrophy that follows a sustained increase in loadupon the heart. Such an increase in workload occurs in the survivingportions of the heart following a severe myocardial infarction.Therefore, pyrimidine nucleosides or acylated derivatives are usefultherapeutic agents in the treatment of or prevention of heart failurefollowing myocardial infarction. There are currently no therapeuticagents in contemporary clinical practice that operate by supporting thebiochemical mechanism underlying myocardial energy metabolism orcapacity for adaptation to sustained increases in workload. Theseresults indicate that such an approach yields significant functionalbenefits.

Example IV

Treatment of Liver Damage With Acylated Pyrimidine Nucleosides

The effect of oral triacetylcytidine and triacetyluridine on chemicallyinduced liver damage was assessed. Chronic treatment of rodents withcarbon tetrachloride is a standard model for inducing a heptatopathythat eventually leads to cirrhosis.

20 male F344 rats (200 g) received injections of carbon tetrachloride(0.2 ml/kg of 50% CCl₄ in corn oil) twice per week for 8 weeks. Afterthe first 2 weeks of treatment with carbon tetrachloride, half of theanimals were subjected to oral administration (gavage) of a mixture oftriacetyluridine (TAU) and triacetylcytidine (TAC) (50 mg/kg of each in1 ml of water, twice per day) for the remaining 6 weeks. The other halfof the animals (controls) received equivalent volumes of water bygavage. At the end of 8 weeks of carbon tetrachloride treatment, thefunctional capacity of the livers was assessed by their capacity toremove bromsulphthalein (BSP) from the circulation (a standard test ofliver function). The rats were anesthetized (ketamine 80 mg/kg andxylazine 13 mg/kg) and their carotic arteries were catheterized for BSPadministration and blood sampling. BSP (50 mg/kg in 0.5 ml saline) wasadministered as a bolus. Blood samples (0.2 ml) were taken periodically,and plasma BSP concentrations were determined by adding 20 microlitersof plasma to 1 ml 0.1 M NaOH and recording UV absorbance at 575 nm.

As is shown in FIG. 11, animals that received TAC and TAU duringtreatment with carbon tetrachloride had a significantly better capacityto remove BSP from their circulation than did the control animals,indicating that TAC and TAu provide significant protection of the liveragainst damage from carbon tetrachloride.

Example V

Preparation of 2′,3′,5′-Tri-O-acyl uridine From acid anhydrides:

To 1 gram of uridine dissolved in 20 ml anhydrous pyridine (previouslydried over potassium hydroxide) is added at room temperature 3.1 molarequivalents of the acid anhydride of the desired acyl compound (e.g.,acetic anhydride, lactate anhydride, butyric anhydride, etc.). Thereaction mixture is then heated to 80-85° C. for 2 hours, cooled, pouredinto ice water, and the esters recovered by extraction three times withequal volumes of chloroform. The chloroform is then washed with ice-cold0.01 N sulfuric acid, 1% aqueous sodium bicarbonate, and finally water.After drying with sodium sulfate, the chloroform is evaporated and theresidual oil or crystals are subjected to chromatography (adapted fromNishizawa et al., Biochem. Pharmacol. 14:1605 (1965).

From acid chlorides:

To 1 gram of uridine in 20 ml anhydrous pyridine is added, at 5° C., 3.1molar equivalents of the acid chloride of the desired acyl compound(e.g., palmitoyl chloride, acetyl chloride, etc.). The mixture is heldat room temperature overnight, added to ice water, and worked up asindicated above (adapted from Nishizawa et al., Biochem. Pharmacol.14:1604 (1965)).

Example VI

Preparation of 5-Acyl Uridine

To 1 gram of uridine dissolved in 20 ml anhydrous pyridine is added, atroom temperature, 1.0 molar equivalent of the acid anhydride of thedesired acyl compound. The reaction is then heated to 80-85° C. for twohours, cooled, poured into ice water, and the esters recovered byextraction three times with equal volumes of chloroform. The chloroformis then washed ice cold 0.01 N sulfuric acid, 1% aqueous sodiumbicarbonate, and finally water. After drying with sodium sulfate, thechloroform is evaporated and the residual oil or crystals are subjectedto chroma tography. The major product, which is isolated by chromatography, is the 5′-substituted ester (adapted from Nishizawa et al.,Biochem. Pharmacol. 14:1605 (1965)).

Alternatively, selective 5′-acylation of uridine may be accomplished bysuspending 1 gram of uridine in 30 ml of 1:1pyridine:N,N-dimethylformamide cooled to 0° C. in an ice bath. 1.0 molarequivalent of the acid chloride of the desired acyl compound is addeddropwise to the mixture, which is stirred at 0° C. for 12-24 hours. 3 mlof water is added, and then the solvents are evaporated in vacuo at 50°C. The residue is dissolved in methanol and adsorbed onto approximately3 grams of silica gel, and the excess solvent is evaporated off. Tolueneis evaporated three times from the solid mass, and the whole is loadedonto a 3×15 cm slurry-packed column of silica gel in chloroform, andeluted with a linear gradient of chloroform (200 ml) to 20:80methanol:chloroform (200 ml). The appropriate fractions, as determinedby TLC, are combined, and the solvents are evaporated to yield thedesired product that is either recrystallized or dried in vacuo to aglass (adapted from Baker et al., J. Med. Chem. 21:1218 (1978)).

Example VII

Preparation of 2′,3′-Diacyl Uridine

To a stirred suspension of 1 gram of uridine in 20 ml dry N,N-dimethylformamide is added 2.4 molar equivalents of imidazole followedby 1.2 molar equivalents of t-butyldimethylchlorosilane. The mixture isstirred with protection from moisture at room temperature for 20 hours,at which time the solvent is removed at 50° C. in vacuo. The residue isdissolved in 15 ml of ethyl acetate, the solution is washed with 10 mlof water, and the extract is dried with magnesium sulfate and evaporatedto give a syrup. Crystallization from 10 ml of hot chloroform, to whichis added hexane to the point of opalescence, followed by slow cooling toroom temperature, gives 5′-(t-butyldimethylsilyl) uridine.

To a stirred suspension of 1 gram of 5′-(t-butyldimethylsilyl) uridinein 15 ml of dry pyridine cooled to 0° C., is added 2.1 molar equivalentsof the appropriate acid anhydride of the desired acyl compound, and themixture is stirred with protection from moisture for 20 hours at 0-5°C., at which time the reaction is terminated by addition of a few ml ofwater. The solvent is evaporated and the residue is dissolved in 15 mlof chloroform, washed with 2×15 ml of saturated sodium hydrogencarbonate, and then the water, dried (magnesium sulfate) and evaporatedto give a thick, clear syrup, which is then dried in vacuo at 25° C.

To a stirred solution of the above acrylated product in 30 ml of drytetrahydrofuran is added 0.2 ml glacial acetic acid, followed by 1.5-2.3grams of tetrabutylammonium fluoride, and the reaction is monitored byTLC (9:1 chloroform methanol). Upon complete removal of thet-butyldimethylsilyl group from the 5′ hydroxyl group of the acylateduridine derivative, the fluoride is removed from the mixture byfiltration through a layer of 30 grams of silica gel, and the productsare eluted with tetrahydrofuran. The crude product, obtained uponevaporation of the solvent is recrystallized from acetone, yielding thedesired 2′3′-diacyl uridine derivative (adapted from Baker et al., J.Med. Chem. 22:273 (1979)).

Example VIII

Preparation of N³,2′,3,5′Tetraacyl Uridine

The acylation of the secondary amine in the 3 position of the pyrimidinering is accomplished by reacting 2′,3′,5′-tri-O-acyl uridine with 1.1molar equivalents of the acid chloride of the desired acyl substituentin an aprotic solvent (such as ether, dioxane, chloroform, ethylacetate, acetonitrile, pyridine, dimethylformamide and the like) in thepresence of 1-5 molar equivalents of an organic base (especiallyaromatic amines such as pyridine, trialkylamines, orN,N-dialkylanilines) adapted from Fujii et al., U.S. Pat. No.4,425,335). The acyl substituent on the secondary amine can be the sameor different from those on the hydroxyl groups of the ribose moiety.

Example IX

Preparation of 2′,3′,5′-Tri-O-acyl Cytidine

One gram of cytidine hydrochloride is dissolved in 10 ml ofN,N-dimethylformamide. 3.1 molar equivalents of the acid chloride isadded and the mixture is stirred overnight at room temperature. Thereaction mixture is concentrated in vacuo to an oil, and triturated with1.1 ethyl acetate: diethyl ester. The oil is then triturated with 1Nsodium hydrogen carbonate. The crystalline solid is collected, washedwith water, dried, and recrystallized (adapted from Gish et al., J. Med.Chem. 14:1159 (1971)).

Example X

Preparation of 5′-Acyl Cytidine

One gram of cytidine hydrochloride is dissolved in 10 ml ofN,N-dimethylformamide. 1.1 molar equivalents of the acid chloride of thedesired acyl substituent is added, and the mixture is stirred overnightat room temperature. The reaction mixture is concentrated in vacuo to anoil, and triturated with 1:1 ethyl acetate: diethyl ether. The oil isthen triturated with 1N sodium hydrogen carbonate. The crystalline solidis collected, washed with water, dried, and recrystallized (adapted fromGish et al., J. Med. Chem. 14:1159 (1971)).

Example XI

Preparation of N⁴-Acyl Cytidine

The N⁴-amino group of cytidine is the best nucleophile among the aminoand hydroxyl functionalities of cytidine. Selective N⁴-acylation can beaccomplished by treating cytidine with appropriate acid anhydrides inpyridine or a mixture of pyridine and N,N-dimethylformamide.Specifically, 1 gram of cytidine is suspended in 80 ml of dry pyridine;1.5 molar equivalents of desired acid anhydride is added, and themixture is refluxed for 2 hours. The solvent is removed in vacuo, andthe resulting white solid is recrystallized from ethanol.

Alternatively, cytidine (1 gram) is dissolved in a mixture comprising70:30 pyridine:N,N-dimethylformamide. 1.5 molar equivalents of the acidanhydride of the desired acyl substituent is added, and the mixture isstirred overnight at room temperature, after which it is poured intowater and stirred. The solvent is removed in vacuo to leave a whitesolid, which is extracted with diethy ether. The residue isrecrystallized from ethanol (adapted from Sasaki et al., Chem. Pharm.Bull 15:894 (1967)).

An alternative procedure is to dissolve cytidine in a mixture of waterand a water-miscible organic solvent (such as dioxane, acetone,acetonitrile, N,N-dimethylformamide, dimethylsulfoxide, tetrahydrofuran,etc.) and to treat that solution with abut a twofold excess of anappropriate acid anhydride. For example, 1 gram of cytidine dissolved in5 ml of water is mixed with 15 to 100 ml dioxane (more dioxane is neededfor more lipophilic substituents), and 2 molar equivalents of the acidanhydride of the desired acyl substituent is added. The mixture isstirred for 5 hours at 80° C. (or 48 hours at room temperature), andthen the solvent is removed in vacuo. The residue is washed with hexaneor benzene, and recrystallized from ethanol or ethyl acetate (adaptedfrom Akiyama et al., Chem. Pharm. Bull. 26:981 (1978).

Example XII

Preparation of N⁴,2′,3′,5′Tetraacyl Cytidine

Compounds in which the acyl substituent of the N⁴ amino group and thehydroxyl groups of the ribose ring of cytidine are the same (e.g.,tetraacetyl cytidine) are prepared by dissolving or suspending cytidinein dry pyridine, adding at least 4 molar equivalents of the acidchloride or acid anhydride of the desired substituent, and stirring themixture overnight at room temperature. The solvent is removed in vacuoand the residue is washed and recrystallized.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters of composition, conditions, and modes ofadministration without departing from the spirit or scope of theinvention or any embodiment thereof.

What is claimed is:
 1. An acyl derivative of uridine having the formula(II)

wherein R₁, R₂, and R₃ are the same or different and each is hydrogen oran acyl radical of (a) an unbranched fatty acid with 5 to 22 carbonatoms, (b) an amino acid selected from the group consisting of glycine,L-forms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine, and ornithine,(c) a dicarboxylic acid of 3 to 22 carbon atoms, or (d) a carboxylicacid selected from one or more of the group consisting of glycolic acid,pyruvic acid, lactic acid, enolpyruvic acid, lipoic acid, pantothenicacid, acetoacetic acid, p-aminobenzoic acid, betahydroxybutyric acid,orotic acid, and creatine, provided that at least one of saidsubstituents R₁, R₂, and R₃ is not hydrogen, and further provided thatif any of said substituents R₁, R₂, and R₃ is hydrogen and if saidremaining substituents are acyl radicals of a straight chain fatty acid,then said straight chain fatty acid has 8 to 22 carbon atoms, or apharmaceutically acceptable salt thereof.
 2. An acyl derivative ofuridine having the formula (I)

wherein R₁, R₂, and R₃ are the same or different and each is hydrogen oran acyl radical of a carboxylic acid selected from one or more of thegroup consisting of a fatty acid of 2 to 22 carbon atoms, glycolic acid,pyruvic acid, lactic acid, enolpyruvic acid, an amino acid, lipoic acid,pantothenic acid, succinic acid, fumaric acid, adipic acid, acetoaceticacid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, andcreatine, and R₄ is an acyl radical of a carboxylic acid selected fromone or more of the group consisting of a fatty acid of 2 to 22 carbonatoms, glycolic acid, pyruvic acid, lactic acid, enolpyruvic acid, andamino acid, lipoic acid, pantothenic acid, succinic acid, fumaric acid,adipic acid, acetoacetic acid, p-aminobenzoic acid, betahydroxybutyricacid, orotic acid, and creatine, or a pharmaceutically acceptable saltthereof.
 3. The acyl derivative of claim 2 wherein said amino acid isselected from the group consisting of glycine, the L forms of alanine,valine, leucine, isoleucine, proline, phenylalanine, tyrosine, cysteine,cystine, methionine, tryptophan, aspartic acid, glutamic acid, arginine,lysine, histidine, ornithine, carnitine, and hydroxylysine.
 4. Acomposition comprising the acyl derivative of claim 1 and apharmaceutically acceptable carrier.
 5. A unit dose of the compositionof claim 4 comprising an amount of said acyl derivative being theequivalent of 10-3000 mg of uridine.
 6. A composition comprising amixture of at least one acyl derivative of claim 1, at least one acylderivative of cytidine selected from the group consisting of2′,3′,5′-tri-O-acetyl cytidine, 2′,3′,5′-tri-O-propionyl cytidine, or2′,3′,5′-tri-O-butyryl cytidine and a pharmaceutically acceptablecarrier.
 7. A unit dose of the composition of claim 6 comprising amountsof said acyl derivatives being the equivalent of 10-3000 mg of uridineand 10-3000 mg of cytidine.
 8. The composition of claim 4 in the form ofa liquid, a suspension, a tablet, a dragee, an injectable solution, or asuppository.
 9. A composition as recited in claim 1 wherein said acylderivative of uridine is 2′,3′,5′-tri-O-acetyl uridine,2′,3′,5′-tri-O-propionyl uridine, or 2′,3′,5′-tri-O-butyryl uridine. 10.A pharmaceutical composition comprising an acyl derivative of uridinehaving the formula (I)

wherein R₁, R₂, R₃ and R₄ are the same or different and each is hydrogenor an acyl radical of a carboxylic acid selected from the groupconsisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvicacid, an amino acid, a fatty acid of 2 to 22 carbon atoms, lipoic acid,pantothenic acid, succinic acid, fumaric acid, adipic acid, acetoaceticacid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, andcreatine, provided that at least one of said R substituents is nothydrogen, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier, wherein said composition is in theform of an oral suspension, tablet, dragee or capsule.
 11. Apharmaceutical composition comprising an acyl derivative of cytidinehaving the formula (III)

wherein R₁, R₂ and R₃ are the same or different and each is hydrogen oran acyl radical of a carboxylic acid selected from the group consistingof glycolic acid, pyruvic acid, lactic acid, enolpyruvic acid, an aminoacid, a fatty acid of 2 to 22 carbon atoms, lipoic acid, pantothenicacid, succinic acid, fumaric acid, adipic acid, acetoacetic acid,p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and creatine,provided that at least one of said R substituents is not hydrogen, andR₄ is an amino acid, or a pharmaceutically acceptable salt thereof, anda pharmaceutically acceptable carrier, wherein said composition is inthe form of an oral suspension, tablet, dragee or capsule.
 12. Acomposition as in claim 10 or 11 wherein said pharmaceuticallyacceptable carrier is a filler selected from the group consisting of asugar, a cellulose preparation and a calcium phosphate.
 13. Acomposition as in claim 12 wherein said sugar is lactose, sucrose,mannitol or sorbitol.
 14. A composition as in claim 10 or 11 whereinsaid pharmaceutically acceptable carrier is a binder selected from thegroup consisting of maize starch, wheat starch, rice starch, potatostarch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethyl cellulose, and polyvinyl pyrrolidone.15. A composition as in claim 10 or 11 wherein said pharmaceuticallyacceptable carrier is selected from the group consisting ofcarboxymethylstarch, cross-linked polyvinyl pyrrolidone, agar, oralginic acid or a salt thereof.
 16. A composition as in claim 10 or 11wherein said pharmaceutically acceptable carrier is a flow-regulatingagent or lubricant selected from the group consisting of silica, talc,stearic acid or salts thereof, and polyethylene glycol.
 17. Acomposition as in claim 16 wherein said salt is magnesium stearate orcalcium stearate.
 18. A composition as in claim 10 or 11 wherein saidpharmaceutically acceptable carrier is a coating selected from the groupconsisting of sugar solutions which optionally contain gum arabic, talc,polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide,lacquer solutions, and a cellulose preparation.
 19. A composition as inclaim 18 wherein said cellulose preparation is acetylcellulose phthalateor hydroxylpropylmethylcellulose phthalate.
 20. A composition as inclaim 10 or 11 wherein said pharmaceutically acceptable carrier isgelatin.
 21. A composition as in claim 10 or 11 wherein saidpharmaceutically acceptable carrier is a base selected from the groupconsisting of triglycerides, paraffin hydrocarbons, polyethylene glycolsand higher alkanols.
 22. A composition as in claim 10 or 11 wherein saidpharmaceutically acceptable carrier is a lipophilic solvent or vehicleselected from the group consisting of fatty oils and fatty acid esters.23. A composition as in claim 10 or 11 wherein said pharmaceuticallyacceptable carrier is an aqueous injection suspension selected from thegroup consisting of sodium carboxymethylcellulose, sorbitol, anddextran.